A research program is proposed on the induction and consequences of protein synthesis errors in eukaryotic cells. This program is based on findings made in our laboratory, concerning the stimulation of misreading by paromomycin, and certain other structurally related members of the aminoglycoside antibiotics. We propose to define the chemical structure necessary for the misreading activity and to show that these molecules act at the level of the ribosome. These studies will be complemented by measurements of the binding of paromomycin to eukaryotic ribosomes. In addition, studies on misreading in eukaryotic systems will be extended to other classes of antibiotics, structurally distinct from the aminoglycosides. Our work has shown that the ciliated protozoan, Tetrahymena, is very sensitive to paromomycin; the drug seems to cause translation errors in the intact organism. We propose to characterize a paromomycin-resistant mutant that we have isolated in order to establish the biochemical basis for drug resistance. Also, we propose to attempt the isolation of paromomycin-dependent mutants as a part of a long-term strategy to develop a system of nonsense mutations and nonsense suppressors to aid in the analysis of the genetics of a lower eukaryote. The protozoan responds to paromomycin and other agents that generate faulty polypeptide with a dramatic induction of new proteins. We wish to explore the biochemistry of this response, which may prove to be useful as a model system for the study of gene regulation in a lower eukaryote. Finally, we propose to test whether an induction of proteolytic enzymes occurs as a part of the response, and to examine if those enzymes form part of a system to protect the organism from deleterious effects of errors during protein synthesis.